Liposome-Supplemented Cell-free Synthesis of a Peptide that Forms β-barrel Nanopores

Rational design of nanopore-forming proteins and peptides is a promising approach for single-molecule analysis by using custom-designed nanopores tailored to specific target molecules. One challenge in this approach is the expression or synthesis of the designed nanopores, due to their high hydrophobicity. We sought to streamline the synthesis of an artificial and highly aggregative nanopore-forming peptide using liposome-supplemented cell-free protein synthesis. By optimizing the liposomal lipid composition, we recovered yields of the expressed aggregative peptide that were suitable for functional measurements. Channel current measurements involving peptide-embedded liposome fusion to a planar lipid bilayer confirmed the pore formation and single-molecule detection capabilities of the expressed peptide nanopore. Through the exploration of various lipid compositions, it was found that the incorporation of anionic lipids and a sufficient acyl chain length were crucial for efficient peptide insertion and the reproduction of native oligomeric states. Such modulation of liposomal lipid composition alongside cell-free protein synthesis has the capacity to alleviate the aggregation propensity constraints in the design of nanopores and other membranous peptides.